Candidate: Sarita Patil, MD is a staff physician in Allergy and Immunology at Massachusetts General Hospital (MGH) and a junior investigator as an Instructor in Medicine at Harvard Medical School (HMS). During her fellowship, she conducted translational research, which led to the development of a fluorescent allergen- specific B cell tetramer that could successfully identify peanut-specific B cells in the periphery f peanut- allergic patients undergoing immunotherapy. Building on that technology, this K23 application aims to understand the role of the humoral response in oral food immunotherapy, specifically investigating the immunoglobulin response to the peanut allergen Ara h 2 on a clonal level, allowing for greater insight into mechanisms of immunotherapy in human patients with food allergy. The proposed project combines research in clinical trials, basic science, and bioinformatics to develop a unique expertise in translational research. The work, training activities, and mentoring plan outlined in this proposal will successfully position Dr. Patil for hr first R01 application and an independent career as a physician-scientist. Mentorship, Training Activities, and Environment: Dr. Patil will perform the proposed project in the Center for Immunology and Inflammatory Diseases (CIID) at MGH under the mentorship of Dr. Wayne Shreffler and at Massachusetts Institute of Technology (MIT) under the co-mentorship of Dr. J. Christopher Love. The CIID is a multidisciplinary basic science center that serves as the foundation of immunology research at MGH. In addition, Dr. Robert Anthony at MGH and Dr. Steven Kleinstein at Yale School of Medicine will serve on Dr. Patil's Advisory Committee to provide expertise on immunoglobulin glycosylation in allergic diseases as well as immunoglobulin repertoire analysis using computational biology approaches, respectively. To complement her proposed projects and the expertise of her mentors, Dr. Patil will complete didactic courses in translational research, statistics, sequencing, proteomics, and bioinformatics through the Harvard School of Public Health and the Harvard Catalyst. The collaborative opportunities, intellectual environment and resources available to Dr. Patil are outstanding. Research: IgE-mediated peanut allergy poses a significant public health risk, as minute peanut exposures can cause severe allergic reactions. Clinical trials of peanut oral immunotherapy (PNOIT) with gradual incremental peanut exposure under careful observation in children have been successful in inducing desensitization; however, only a fraction of these children have long-lived tolerance. Patients undergoing PNOIT often have a decrease in their peanut-specific IgE and a gradual increase in their peanut-specific IgG and IgG4 levels, but the mechanistic contribution of these changes in peanut-specific immunoglobulins to long-term tolerance is still unknown. This project addresses how PNOIT affects the B cell repertoire and how those changes correlate to the heterogeneous clinical outcomes that are expected. We hypothesize that long-lasting clinical tolerance following PNOIT directly correlates with, and is partially dueto the induction of high affinity and oligoclonal allergen-specific functional antibodies. Using a fluorescent tetramer-based approach for the identification of allergen-specific B cells, we hope to identify clinical prognostic markers, and improve our understanding of the mechanism of immunotherapy, specifically the pathophysiological role of antibodies and their potential as both biomarkers of tolerance and therapeutic agents. To do so, we propose mapping the allergen-specific B cell repertoire using proteomics and next- generation sequencing in PNOIT patients. We also propose functional studies of recombinant antibodies from the allergen-specific B cells to explore the underlying mechanism of tolerance in PNOIT. By studying both peanut allergic patients who develop variable tolerance during PNOIT as well as peanut allergic patients who naturally outgrew their allergies, we hope to elucidate the contribution of the B cell repertoire t the induction of long-lived tolerance in peanut allergy. These efforts may help identify relevant clinical biomarkers and potential therapeutic avenues for the improvement of PNOIT for the treatment of peanut allergy.